The embryos generated from pairwise breeding of single gene and multi-gene heterozygote mutant fish were grown to adulthood (3–6 months). Fin clips from adult fish were processed for DNA extraction using the “Extract-N-Amp” kit (Sigma-Aldrich) and used for genotyping by fluorescent PCR method as described [67 (link)]. The genotyping data were used to analyze for Mendelian ratios of surviving homozygous knockout fish compared to the homozygote WT and heterozygous fish. Under the null hypothesis of no viability selection, progeny genotypes should conform to an expected Mendelian ratio of 1:2:1. Deviations from expected number of homozygous knockouts (25%) were tested with goodness-of-fit Chi-square statistical analysis. If the parent fish were heterozygote for mutations in more than one gene, data were analyzed for survival of all possible genotypes expected from these breeding. To get sufficient number of fish genotyped, we analyzed progenies from two breeding for most alleles. To determine the presence of both sexes among surviving adults, all genotyped fish were categorized as males and females and counted.
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